Molecular cloning and nucleotide sequence of the 3-isopropylmalate dehydrogenase gene of Candida utilis

A 3-isopropylmalate dehydrogenase (3-IMDH, EC 1.1.1.85) gene was cloned from a gene library of Candida utilis. One of the plasmids, pYKL30, could complement Escherichia coli leuB and Saccharomyces cerevisiae leu2 auxotrophs; a 2.2 kb HindIII fragment subcloned in pBR322 could still complement the leuB mutation. Southern hybridization confirmed that this fragment was derived from C. utilis. An open reading frame of 1089 bp that corresponded to a polypeptide of 363 amino acids, one residue shorter than the 3-IMDH of S. cerevisiae, was found in the cloned fragment. The homology between the 3-IMDHs of C. utilis and S. cerevisiae was 76.2% in nucleotides and 85.4% in amino acids. In contrast, the homology between the 3-IMDHs of C. utilis and Thermus thermophilus was much smaller and was restricted to some regions of the gene.     

Fusion of the Saccharomyces cerevisiae leu2 gene to an Escherichia coli beta-galactosidase gene.

The promoter and translation initiation region of the Saccharomyces cerevisiae leu2 gene was fused to the Escherichia coli beta-galactosidase gene. This fusion located the control region of the leu gene and orientated its direction of expression. When the fusion was placed into yeast cells, beta-galactosidase was expressed under the same regulatory pattern as the original leu2 gene product: its synthesis was repressed in the presence of leucine and threonine. Sensitive chromogenic substrates for beta-galactosidase were used to detect expression in isolated colonies growing on agar medium. Mutant yeast cells with increased beta-galactosidase activity were identified by the color of the colonies they formed. One class of mutants obtained appeared to affect ars1 plasmid maintenance, and another class appeared to affect beta-galactoside uptake.     

Functional expression of two Bacillus subtilis chromosomal genes in Escherichia coli.

EcoRI-cleaved deoxyribonucleic acid segments carrying two genes from Bacillus subtilis, pyr and leu, have been cloned in Escherichia coli by insertion into a derivative of the E. coli bacteriophage lambda. Lysogenization of pyrimidine- and leucine-requiring auxotrophs of E. coli by the hybrid phages exhibited prototrophic phenotypes, suggesting the expression of B. subtilis genes in E. coli. Upon induction, these lysogens produced lysates capable of transducing E. coli pyr and leu auxotrophs to prototrophy with high frequency. Isolated DNAs of these bacteriophages have the ability to transform B. subtilis auxotrophs to pyr and leu independence and contain EcoRI-cleaved segments which hybridize to corresponding segments of B. subtilis.     

Development of a transformation system for the flavinogenic yeast Candida famata

Riboflavin-overproducing mutants of the flavinogenic yeast Candida famata are used for industrial riboflavin production. This paper describes the development of an efficient transformation system for this species. Leucine-deficient mutants have been isolated from C. famata VKM Y-9 wild-type strain. Among them leu2 mutants were identified by transformation to leucine prototrophy with plasmids YEp13 and PRpL2 carrying the Saccharomyces cerevisiae LEU2 gene. DNA fragments (called CfARSs) conferring increased transformation frequencies and extrachromosomal replication were isolated from a C. famata gene library constructed on the integrative vector containing the S. cerevisiae LEU2 gene as a selective marker. The smallest cloned fragment (CfARS16) has been sequenced. This one had high adenine plus thymine (A+T) base pair content and a sequence homologous to the S. cerevisiae ARS Consensus Sequence. Methods for spheroplast transformation and electrotransformation of the yeast C. famata were optimized. They conferred high transformation frequencies (up to 10(5) transformants per microg DNA) with a C. famata leu2 mutant using replicative plasmids containing the S. cerevisiae LEU2 gene as a selective marker. Riboflavin-deficient mutants were isolated from the C. famata leu2 strain and their biochemical identification was carried out. Using the developed transformation system, several C. famata genomic fragments complementing mutations of structural genes for riboflavin biosynthesis (coding for GTP cyclohydrolase, reductase, dihydroxybutanone phosphate synthase and riboflavin synthase, respectively) have been cloned.     

Symbiotic characterization of isoleucine+valine and leucine auxotrophs of Sinorhizobium meliloti

Ten isoleucine+valine and three leucine auxotrophs of Sinorhizobium meliloti Rmd201 were obtained by random mutagenesis with transposon Tn5 followed by screening of Tn5 derivatives on minimal medium supplemented with modified Holliday pools. Based on intermediate feeding, intermediate accumulation and cross-feeding studies, isoleucine+valine and leucine auxotrophs were designated as ilvB/ilvG, ilvC and ilvD, and leuC/leuD and leuB mutants, respectively. Symbiotic properties of all ilvD mutants with alfalfa plants were similar to those of the parental strain. The ilvB/ilvG and ilvC mutants were Nod-. Inoculation of alfalfa plants with ilvB/ilvG mutant did not result in root hair curling and infection thread formation. The ilvC mutants were capable of curling root hairs but did not induce infection thread formation. All leucine auxotrophs were Nod+ Fix-. Supplementation of leucine to the plant nutrient medium did not restore symbiotic effectiveness to the auxotrophs. Histological studies revealed that the nodules induced by the leucine auxotrophs did not develop fully like those induced by the parental strain. The nodules induced by leuB mutants were structurally more advanced than the leuC/leuD mutant induced nodules. These results indicate that ilvB/ilvG, ilvC and one or two leu genes of S. meliloti may have a role in symbiosis. The position of ilv genes on the chromosomal map of S. meliloti was found to be near ade-15 marker.     

Insertion of transposon Tn9 into the spinal (Escherichia coli-Saccharomyces cerevisiae) plasmids and the expression of the prokaryotic gene of chloramphenicol resistance in yeast cells

Transposon Tn9 carrying camr gene which controls resistance to chloramphenicol has been introduced in vivo (in cells of Escherichia coli) into two chimeric shuttle plasmids pYF91 and YEp13. These plasmids consist of the different parts of the E. coli plasmid pBR322, the yeast 2mkm DNA plasmid and the yeast LEU2 structural gene. The plasmidis able to autonomously replicate in both yeast and bacterial cells. A recipient yeast strain carrying cams and leu2 markers was constructed to study the functional expression of the prokaryotic camr gene in eukaryotic yeast cells. The chimeric plasmids pYF91::Tn9 and YEp13::Tn9 were introduced into the yeast and bacterial recipient strains by transformation. The camr LEU2 yeast transformants were isolated. They were genetically unstable when grown on non-selective medium and they simultaneously lost camr and LEU2 markers with a frequency of 10 to 30%. The E. coli transformants were genetically stable under nonselective conditions and they maintain all plasmid markers. The chimeric plasmid pYF91::Tn9 was isolated from the yeast transformants and reintroduced into the cams leuB bacterial strain by transformation. The camr LEUB transformants were obtained. All these data confirm the possibility of the expression of the prokaryotic camr gene in yeast cells and present evidence for introduction of transposon Tn9 into chimeric plasmids.     

Molecular cloning and characterization of supQ/newD, a gene substitution system for the leuD gene of Salmonella typhimurium.

The isopropylmalate isomerase of Salmonella typhimurium and Escherichia coli is a complex of the leuC and leuD gene products. The supQ/new D gene substitution system in S. typhimurium restores leucine prototrophy to leuD mutants of S. typhimurium. Previous genetic evidence supports a model that indicates the replacement of the missing LeuD polypeptide by the newD gene product. This model proposed that this gene substitution is possible when a mutation at the supQ locus (near newD) liberates unaltered newD polypeptide from its normal complex with the supQ protein product. In this study, recombinant plasmids carrying newD, supQ, or both were transformed into E. coli and S. typhimurium strains deleted for the leuD and supQ genes to test the supQ/newD gene substitution model for suppression of leucine auxotrophy. It was determined that the newD gene encodes a 22-kilodalton polypeptide which can restore leucine prototrophy to leuD deletion strains and that a functional supQ gene prevents this suppression. It was also determined that the supQ and newD genes are separated by a gene encoding a 50-kilodalton protein, pB. While there is extensive DNA sequence homology between the leucine operons of S. typhimurium and E. coli, DNA hybridization experiments did not indicate substantial homology between the newD and leuD genes. These data, taken together with previously obtained genetic data, eliminate the possibility that supQ and newD are recently translocated segments of the leucine operon.     

Cloning and expression of the 3-isopropylmalate dehydrogenase gene from Candida albicans

Abstract A variety of Saccharomyces cerevisiae genes e.g. HIS3, LEU2, TRP1, URA3 , are expressed in Escherichia coli and have been isolated by complementation of mutations in the corresponding E. coli genes [1]. The LEU2 gene was one of the first S. cerevisiae genes to be isolated in this way [2], and its isolation led to the development of transformation systems for S. cerevisiae [3,4]. The leuB gene in E. coli [5] and the LEU2 gene in S. cerevisiae [6] both code for 3-isopropylmalate dehydrogenase (3-IMDH; EC 1.1.1.85) which is essential for the biosynthesis of leucine in both organisms. This paper describes the cloning of a fragment of C. albicans DNA carrying the gene for 3-IMDH which will be useful in the development of transformation methods in C. albicans .     

Nonsense suppression in Schizosaccharomyces pombe: the S. pombe Sup3-e tRNASerUGA gene is active in S. cerevisiae

Summary The gene encoding the efficient UGA suppressor sup3-e of Schizosaccharomyces pombe was isolated by in vivo transformation of Saccharomyces cerevisiae UGA mutants with S. pombe sup3-e DNA. DNA from a clone bank of EcoRI fragments from a S. pombe sup3-e strain in the hybrid yeast vector YRp17 was used to transform the S. cerevisiae multiple auxotroph his4-260 leu2-2 trp1-1 to prototrophy. Transformants were isolated at a low frequency; they lost the ability to grow in minimal medium after passaging in non-selective media. This suggested the presence of the suppressor gene on the non-integrative plasmid. Plasmid DNA, isolated from the transformed S. cerevisiae cells and subsequently amplified in E. coli, transformed S. cerevisiae his4-260 leu2-2 trp1-1 to prototrophy. In this way a 2.4 kb S. pombe DNA fragment carrying the sup3-e gene was isolated. Sequence analysis revealed the presence of two tRNA coding regions separated by a spacer of only seven nucleotides. The sup3-e tRNA _Ser ^UGA tRNA gene is followed by a sequence coding for the initiator tRNA^Met. The transformation results demonstrate that the cloned S. pombe UGA suppressor is active in S. cerevisiae UGA mutant strains.     

Amplification of the riboflavin operon genes of Bacillus subtilis in Escherichia coli cells]

Amplification of Bacillus subtilis DNA fragments was performed in Escherichia coli using plasmid RSF2124. The main principle of isolation and cloning hybrid plasmids was described using genes of riboflavin operon as a model. Bac. subtilis DNA was treated with restriction endonuclease EcoR; followed by the agarose gel electrophoretic separation of the resulting fragments. Gels were sliced, DNA was eluted from the corresponding slices and used to transform Bac. subtilis auxotrophs rib A72, rib S110 and rib D107. DNA fraction with the molecular weight 7 . 10(6) daltons restored prototrophy of these mutants. DNA of this fraction was ligated with EcoRI treated plasmid RSF2124 DNA and used for transformation of E. coli rk-mk+. Ampicillin resistant transformants which had lost the colicin production ability, were selected. The presence of riboflavin genes within the hybrid plasmids was detected by transformation of B. subtilis auxotrophs. Three hybrid plasmids (pPR1, pPR2 and pPR3), containing a fragment of Bac. subtilis DNA with the molecular weight 6.8 . 10(-6) daltons including riboflavin operon, were selected. The analysis of the transformation activity of Bac. subtilis DNA and plasmid pPR1 DNA revealed, that there was no restriction activity of Bac. subtilis cells against plasmid DNA amplified in E. coli. Heteroduplex analysis has shown that plasmids pPR1 and pPR2 differ in the orientation of Bac. subtilis DNA fragment. DNA of these plasmids restored prototrophy of the several studied E. coli riboflavin auxotrophs.     

Cloning of a yeast gene coding for the glutamate synthase small subunit (GUS2) by complementation of Saccharomyces cerevisiae and Escherichia coli glutamate auxotrophs

A Saccharomyces cerevisiae glutamate auxotroph, lacking NADP-glutamate dehydrogenase (NADP-GDH) and glutamate synthase (GOGAT) activities, was complemented with a yeast genomic library. Clones were obtained which still lacked NADP-GDH but showed GOGAT activity. Northern analysis revealed that the DNA fragment present in the complementing plasmids coded for a 1.5kb mRNA. Since the only GOGAT enzyme so far purified from S. cerevisiae is made up of a small and a large subunit, the size of the mRNA suggested that the cloned DNA fragment could code for the GOGAT small subunit. Plasmids were purified and used to transform Escherichia coli glutamate auxotrophs. Transformants were only recovered when the recipient strain was an E. coli GDH-less mutant lacking the small GOGAT subunit. These data show that we have cloned the structural gene coding for the yeast small subunit (GUS2). Evidence is also presented indicating that the GOGAT enzyme which is synthesized in the E. coli transformants is a hybrid comprising the large E. coli subunit and the small S. cerevisiae subunit.     

Cloning and physical mapping of the ADE1 gene of the yeast Saccharomyces cerevisiae

ADE1 gene of Saccharomyces cerevisiae codes for the primary structure of SAICAR-synthetase. Mutational changes of ADE1 gene result in the accumulation of red pigment in cells. Colour differences, thus, serve as a basis for the selection of mutants or transformants. ADE1 gene was cloned as a 4.0 kb HindIII fragment of yeast DNA in a shuttle vector by complementing the ade1 mutation in yeast. The study of ADE1 gene expression in Escherichia coli showed that the 4.0 kb fragment containing the ADE1 gene does not complement purC mutations in E. coli. However, prototrophic colonies appeared at a frequency of 10(-7)-10(-8) after incubating clones bearing the recombinant plasmid with ADE1 gene on selective media. The plasmid DNA isolated from such clones complements the purC mutation in E. coli and the ade1 mutation in S. cerevisiae. Structural analysis of the plasmid demonstrated that the cloned DNA fragment contained an additional insertion of the bacterial origin. Further restriction enzyme analysis proved the insertion to be the bacterial element IS1. Expression of the cloned ADE1 gene in S. cerevisiae is controlled by its own promoter, whereas in E. coli it is controlled by the IS1 bacterial element.     

Chimeric plasmids for cloning of deoxyribonucleic acid sequences in Saccharomyces cerevisiae.

Two sets of plasmids, each carrying a Saccharomyces cerevisiae gene and a portion or all of the yeast 2-micron circle linked to the Escherichia coli plasmid pBR322, have been constructed. One of these sets contains a BamHI fragment of S. cerevisiae deoxyribonucleic acid that includes the yeast his3 gene, whereas the other set contains a BamHI fragment of S. cerevisiae that includes the yeast leu2 gene. All plasmids transform S. cerevisiae and E. coli with a high frequency, possess unique restriction endonuclease sites, and are retrievable from both host organisms. Plasmids carrying the 2.4-megadalton EcoRI fragment of the 2-micron circle transform yeast with 2- to 10-fold greater frequency than those carrying the 1.5-megadalton EcoRI fragment of the 2-micron circle. Restriction endonuclease analysis of plasmics retrieved from S. cerevisiae transformed with plasmics carrying the 2.4-megadalton EcoRI fragment showed that in 13 of 96 cases the original plasmic has acquired an additional copy of the 2-mcron circle. These altered plasmids appear to have arisen by means of an interplasmid recombination event while in S. cerevisiae. A clone bank of S. cerevisiae genes based upon one of these composite plasmids has been constructed. By using this bank and selecting directly in S. cerevisiae, the ura3, tyr1, and met2 genes have been cloned.     

Construction of two new vectors for transformation of laboratory,natural and industrial<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains to trifluoroleucine and G418 resistance

Two new plasmids, pEC3 and pECkan, were constructed and their use in yeast transformation described. Both plasmids are derivative of the pRS416 vector, in which the URA3 auxotrophic marker was replaced by the LEU4* gene (pEC3) or the kanMX4 gene (pECkan). pEC3 and pECkan plasmids transformed natural and commercial Saccharomyces cerevisiae strains to 5,5,5-trifluoro-DL-leucine and G418 (aminoglycoside related to gentamicin) resistance, respectively, with efficiency ranging from 10(-5) to 10(-7) transformants per number of viable cells. pEC3 transformed the Leu- laboratory strain, carrying the mutations leu4 leu9, to leucine prototrophy with efficiency of approximately 10(-4).     

Expression of the cloned uracil permease gene of Saccharomyces cerevisiae in a heterologous membrane

A piece of DNA of the yeast Saccharomyces cerevisiae complementing the uracil permease gene was introduced into a plasmid able to replicate autonomously in Schizosaccharomyces pombe. A strain of S. pombe lacking uracil transport activity was transformed with this new plasmid carrying the gene of S. cerevisiae. The behaviour of the transformant shows not only an expression of the uracil permease gene in the heterologous membrane but also that the transport of uracil is active and coupled to the energy furnishing system of the heterologous host.     

The ADE2 gene from Saccharomyces cerevisiae: sequence and new vectors

We have determined the sequence of a DNA fragment encoding the ADE2 gene from Saccharomyces cerevisiae. A DNA fragment of 2241 bp capable of complementing ade2 mutations was modified so it is available as a single BglII fragment for use in yeast vectors or for gene disruptions. The minimal fragment codes for a putative protein which is highly similar to the protein encoded by the ADE6 gene from Schizosaccharomyces pombe and to the proteins encoded by the purEK operon of Escherichia coli.     

Isolation and study of hybrid plasmids carrying Escherichia coli threonine operon genes

A fragment of Escherichia coli chromosome containing the intact threonine operon or its distinct genes has been cloned on the pBR322 plasmid. This fragment has been mapped using some restriction endonucleases. Cloning results in an increased level of appropriate enzyme activity in cells containing hybrid plasmids. Those carrying the complete threonine operon are capable of accumulating threonine up to 5 g/l in culture medium during 48 h. When multi-copy plasmids are used for gene cloning, interpretation of experiments aimed at transformation of auxotrophic bacterial strains, might be complicated. For example, transformation of appropriate threonine auxotrophs by a hybrid plasmid carrying mutation in the threonine gene, might result in prototrophic phenotype. It is possible that the great amount of mutant enzyme molecules compensated their low activity. On the contrary, the presence of a gene within the plasmid, as shown by restriction and biochemical analysis, did not always ensure the growth on a minimal medium of auxotrophs transformed by this plasmid.     

Expression of thymidylate synthetase activity in Bacillus subtilis upon integration of a cloned gene from Escherichia coli

The gene from Escherichia coli encoding thymidylate synthetase was cloned in the plasmid pBR322. The resulting chimeric plasmid, pER2, was effective in transforming both E. coli and Bacillus subtilis to thymine prototrophy. Uncloned linear E. coli chromosomal DNA was unable to transform thymine-requiring strains of B. subtilis to thymine independence. Linearization of the chimeric plasmid, pER2, with restriction enzymes markedly diminished its ability to transform B. subtilis auxotrophs. The Thy+ transformants derived from the transformation of B. subtilis with pER2 DNA did not contain detectable extrachromosomal DNA as demonstrated by Southern hybridization patterns and centrifugation in CsCl gradients of DNA isolated from B. subtilis colonies transformed with the chimeric plasmid. We conclude that the DNA from the chimeric plasmid was integrated into the chromosome of B. subtilis, demonstrating that extensive homology is not required for the integration of foreign DNA. This is the first reported case of a gene from a Gram-negative bacterium functioning in a Gram-positive organism.     

Construction of an effective host-vector system for the yeast Saccharomyces exiguus Yp74L-3.

An effective host-vector system specific to the yeast Saccharomyces exiguus Yp74L-3 was constructed to promote the molecular genetic analyses for the yeast. To obtain a stable reversionless host strain, we constructed an S. exiguus strain carrying leu2::ScURA3 by disrupting the S. exiguus LEU2 gene with the S. cerevisiae URA3 gene. A vector plasmid unique to S. exiguus was subsequently developed by inserting both the LEU2 gene and an ARS cloned from S. exiguus into an Escherichia coli phagemid, pUC119. The vector constructed, pTH119 was able to transform the S. exiguus leu2::ScURA3 strain to Leu+ efficiently. The stability of the vector in the S. exiguus host cells resembled that of a YRp-type vector in S. cerevisiae.     

Large-scale production of yeast hybrids by electrofusion

Abstract Large-scale production of electrically fused yeast protoplasts from Saccharomyces cerevisiae AH 22[pADH 040-2] and S. cerevisiae AH215 was achieved by the use of the so-called helical fusion chamber. Both strains were of the same mating type a and carried the following auxotrophic markers: his4 in the case of AH22 and leu2, his3 in the case of AH215.
AH22 also is a carrier of the plasmid pADH 040-2. This plasmid confers the leu2 gene of yeast and the β-lactamase gene from Escherichia coli , and this feature enables quick detection of plasmid-positive cells.
After dielectrophoresis (275 V/cm, 800 kHz) fusion was induced by two field pulses (10 kV/cm, 10 μs duration) applied at an interval of 0.5 s. 50 to 60 hybrids per run were isolated after regeneration on selection medium.